1. Field of the Invention
The present invention relates to the field of assays for measuring the intracellular movement (“trafficking”) of proteins containing at least one transmembrane domain, such as a cell surface receptor.
2. Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Protein synthesis and its processing are highly regulated events done in a tightly scrutinized and controlled manner at the transcriptional, translational and post-translational levels involving the endoplasmic reticulum (ER), Golgi apparatus, the plasma membrane, endosome, phagosome and lysosome. Protein synthesis and its folding occur in endoplasmic reticulum. The proteins adopt distinct conformations and mature before reaching their site of action. The process involves strict quality control mechanisms that ensure that improperly/misfolded proteins are accumulated in the ER and are later degraded via the proteosome pathway. In this manner, only the preciously folded proteins are allowed to exit ER and follow the maturation pathway before reaching their site of action.
Trans-membrane proteins such as GPCR's are a part of large family of cell-surface receptors and central to present day drug discovery research. All GPCR's share some unique features of having an extracellular N-terminal fragment, seven trans-membrane domains forming a trans-membrane core, three exoloops, three cytoloops and an intracellular C-terminal segment. However, the different sections vary in size, an indication of their diverse structures and functions. (Attwood T K, Findlay J B, 1994, Fingerprinting G-protein coupled receptors, Protein Eng. 7 (2): 195-203; Kolakowski L F Jr, 1994 GCRDb: a G-protein-coupled receptor database, Receptors Channels 2 (1): 1-7; Foord S M, Bonner T I, Neubig R R, Rosser E M, Pin J P, Davenport A P, Spedding M, Harmar A J, 2005, International Union of Pharmacology. XLVI. G protein-coupled receptor list, Pharmacol Rev 57 (2): 279-88, InterPro). GPCR's broadly can be grouped into six classes based on sequence homology and functional similarity, as follows.
Class A(Rhodopsin-like)Class B(Secretin receptor family)Class C(Metabotropic glutamate/pheromone)Class D(Fungal mating pheromone receptors)Class E(Cyclic AMP receptors)Class F(Frizzled/Smoothened)
A GPCR adopts a tertiary structure with the seven trans-membrane helices which forms a cavity within the plasma membrane and the cavity serves as a ligand-binding domain. Another common structural feature amongst GPCR's is palmitoylation of one or more sites of the C-terminal tail or the intracellular loops which has the effect of targeting the receptor to cholesterol and sphingolipid-rich microdomains of the plasma membrane called lipid rafts and have a role to participate in rapid receptor signaling.
Ion channels represent another class of membrane protein complexes that play an important function of facilitating the diffusion of ions across the biological membranes. They act as electrical insulators and provide a high conducting, hydrophilic pathway across the hydrophobic interior of the membrane. Their mode of action is highly gated and they switch their confirmations between closed and open states. Depending on the chemical and physical modulators that control the gating activity-ion channels can be classified into the following groups:
1. Ligand-gated channels
2. Voltage-gated channels
3. Second-messenger gated channels
4. Mechanosensitive channels
5. Gap junctions
There are a number of human disorders that can result from misfolded/mutated protein ion channels. For example: Inherited long QT syndrome (LQT), which can cause failure of normal inactivation to increase late Na+ current and prolong the action potential. A number of LQT2-linked mutations have been identified in hERG channels. A common mechanism that has emerged and been linked to LQT2 diseases involves protein trafficking defects which reduce the delivery of channels to the cell membrane. After synthesis and core-glycosylations in ER, hERG protein is exported to the Golgi apparatus for complex glycosylation, sorting and eventual insertion into the surface membrane. Once in Golgi apparatus, hERG channels undergo complex glycosylation. A number of biological functions have been suggestive to be a result of core- and complex glycosylation, including promoting proper protein folding, ER export and regulating protein stability.
Therefore, monitoring the activation and/or inhibition of the trafficking can lead to dramatic cellular effects and will help in elucidating the role of trans-membrane proteins in their normal physiological functionality. To develop the therapies and drugs potentially useful in regulating trafficking in healthy and disease states and to understand fate of a protein through the trafficking pathway depends on monitoring the progression of the protein at different stages in cell.